Photochemical covalent binding of urocanic acid to polynucleic acids

Photolysis of E-[ring-2-14C]urocanic acid (UA) with native or denatured calf thymus DNA leads to covalent binding of the radiolabel to the nucleic acid. A similar observation is made upon photolysis of the labeled UA with the polyribonucleotides, in which case a strong preference is observed for binding to poly[U]. DNA or poly[U], which had been reacted with UA and purified by dialysis and multiple precipitations, releases UA upon further irradiation with 254 nm light (as expected for cyclobutane adducts). Quantum efficiencies for binding of the UA to native DNA have been measured at 308 and 266 nm and are 0.30 x 10(-5) and 1.3 x 10(-4), respectively, at comparable reactant concentrations. The large increase at the shorter wavelength (where DNA absorption is more competitive) is taken as evidence for the primary role of a DNA excited state in initiating the binding reaction(s).     

The "cricket bat" flap: a one-stage free forearm flap phalloplasty

Total and subtotal penile reconstruction represents a major surgical challenge. We present a new method and two illustrative cases using a modified design of the radial forearm free-tissue transfer: the "cricket bat" flap.     

A 15N-NMR study of isolated brain in portacaval-shunted rats after acute hyperammonemia.

Acute hyperammonemia was induced by 15NH4+ infusion in portacaval-shunted (PCS) and control rats to investigate its effects on cerebral metabolism of glutamine, glutamate and gamma-aminobutyrate. Cerebral 15N-metabolites were observed by 15N-NMR spectroscopy in the ex vivo brain, removed in toto at the end of infusion. Key 15N-metabolites in the brain and liver were quantitated and their specific activities measured by NMR and biochemical assays in perchloric acid extracts of the freeze-clamped organs. In the ex vivo brain, [gamma-15N]glutamine, present at tissue concentrations of 3-5 mumol/g with 15N enrichment of 36-48%, was observable within 6-13 min of data acquisition. [alpha-15N]glutamine/glutamate, each present at 0.5-1 mumol/g (approx. 10% enrichment), were observed in 27 min. The results demonstrate the feasibility of observing these cerebral metabolites by 15N-NMR within a physiological time scale. In a rat pretreated with glutamine synthetase inhibitor, L-methionine DL-sulfoximine, cerebral [15N]gamma-aminobutyrate was observed after 910 min. In PCS rats, decreased 15NH4+ removal in the liver was accompanied by formation of approx. 2-fold higher concentration of cerebral [gamma-15N]glutamine relative to that in weight-matched controls. The result suggests that increased diffusion of blood-borne 15NH3 into the brain led to increased [gamma-15N]glutamine synthesis in astrocytes as well as ammonia-mediated inhibition of glutaminase.     

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.